Cofactor-dependent specificity of a DEAD-box protein

Abstract

DEAD-box proteins, a large class of RNA-dependent ATPases, regulate all aspects of gene expression and RNA metabolism. They can facilitate dissociation of RNA duplexes and remodeling of RNA-protein complexes, serve as ATP-dependent RNA-binding proteins, or even anneal duplexes. These proteins have highly conserved sequence elements that are contained within two RecA-like domains; consequently, their structures are nearly identical. Furthermore, crystal structures of DEAD-box proteins with bound RNA reveal interactions exclusively between the protein and the RNA backbone. Together, these findings suggest that DEAD-box proteins interact with their substrates in a nonspecific manner, which is confirmed in biochemical experiments. Nevertheless, this contrasts with the need to target these enzymes to specific substrates in vivo. Using the DEAD-box protein Rok1 and its cofactor Rrp5, which both function during maturation of the small ribosomal subunit, we show here that Rrp5 provides specificity to the otherwise nonspecific biochemical activities of the Rok1 DEAD-domain. This finding could reconcile the need for specific substrate binding of some DEAD-box proteins with their nonspecific binding surface and expands the potential roles of cofactors to specificity factors. Identification of helicase cofactors and their RNA substrates could therefore help define the undescribed roles of the 19 DEAD-box proteins that function in ribosome assembly.RNA helicases | annealing D EAD-box proteins are RNA-binding ATPases that are involved in all aspects of RNA metabolism: translation initiation, pre-mRNA splicing, mRNA export and decay, and ribosome biogenesis. In these processes, their functions include RNA duplex unwinding, RNA-protein complex remodeling, RNA duplex annealing, and ATP-dependent RNA binding (1-5).In vivo, these enzymes have specific functions that often involve the recognition of specific RNAs and the discrimination against a myriad of nonsubstrates. Nevertheless, only DbpA, a DEAD-box protein involved in bacterial ribosome assembly (6-8), has sequence-specific ATPase activity (6, 9), which arises from unique sequences in its C terminus (10). Such sequence specificity has not been demonstrated for any other DEAD-box protein and is consistent with their highly conserved structures and the almost universal conservation of residues that contact the RNA. Furthermore, analysis of the structures of RNA-bound DEAD-box proteins reveals that RNA contacts are made with the sugarphosphate backbone (11-17), largely precluding sequence-specific interactions between DEAD-box proteins and RNA.Many DEAD-box and related DEAH-box proteins function in conjunction with cofactors (ref. 18 and references therein). These cofactors can regulate the activity of DEAD-box or DEAH-box proteins by stimulating or inhibiting their ATPase, helicase, RNAbinding, or nucleotide-binding activities. Interestingly, cofactors are often RNA-binding proteins (19)(20)(21)(22)(23)(24)(25). We and others therefore postulated that these cofactors could als...

Mitochondrial ribosome assembly in health and disease

“…Frequently the helicase core is flanked by variable N-and C-terminal extensions that might be critical for the specific function of the individual protein. DEAD-box proteins acquire substrate specificity by interaction with co-factors that target the enzymes to their place of action [110].…”

Stepwise and dynamic assembly of the earliest precursors of small ribosomal subunits in yeast

“…The assembly point of Rrp7 is consistent with its cross-linking sites in helix E of ES6 and helix h26 (Lin et al 2013). In addition, Rok1 interacts with Rrp5 that assembles one step earlier (Torchet et al 1998;Young et al 2013).…”

A pre-ribosomal RNA interaction network involving snoRNAs and the Rok1 helicase

“…Consistent with their adjacent prerRNA binding sites, Rrp5 has been shown to directly interact with Rok1. Furthermore, Rrp5 is suggested to modulate the activity of Rok1 and to confer target specificity on the RNA helicase (Garcia et al 2012;Young et al 2013). In the case of Rrp7, cross-linking sites in ES6H3 and ES7 that are FIGURE 2.…”

“…As the Rok1 helicase contacts all three RNA sequences involved and is required for pre-ribosomal release of snR30, it may function as a key regulator of this structural rearrangement. Furthermore, both ATP-dependent unwinding and ADP-dependent annealing functions of Rok1 have been described (Garcia et al 2012;Young et al 2013), possibly implicating this helicase in formation of base-pairing interactions between ES6 and ES3 after the release of snR30 from ES6.…”